A composition for regulating autophagy of eukaryotic cells and improving mitochondrial dysfunction, and a preparation method and application thereof

Abstract

The present application relates to a kind of compositions for regulating autophagy of eukaryotic cell, improving mitochondrial dysfunction and its preparation method and application.It includes the following weight parts of raw materials: 10-30 parts of broccoli powder, 10-30 parts of wheat germ microcapsule powder, 20-40 parts of raspberry extract, 20-40 parts of banana powder, 2-6 parts of leucine, 0.1-1 parts of pyrroloquinoline quinone disodium salt.The present application also provides the preparation method of broccoli powder, wheat germ microcapsule powder, raspberry extract and banana powder, and the use of the composition.The composition combines the efficacy of the above 6 ingredients, reasonable compatibility, safe and effective, various components activate mitochondria through regulating protein, PINK1 / Parkin-dependent pathway and PINK1 / Parkin-independent pathway;At the same time, by activating PGC-1 alpha signal pathway, promote mitochondrial DNA replication and new mitochondria generation;Stabilize mitochondrial membrane potential, improve ATP synthesis efficiency;Synergistically activate mitochondrial autophagy, improve mitochondrial dysfunction and then relieve aging.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a composition based on broccoli pollen, wheat germ powder, raspberry extract, banana powder, leucine and disodium pyrroloquinoline quinone, and the application of this composition in the preparation of functional foods, pharmaceuticals or health products that regulate eukaryotic autophagy, improve mitochondria and have anti-aging effects. Background Technology

[0002] Mitochondria, as the cell's "energy factories," play a crucial role in cellular respiration and energy production. They provide ATP to cells through oxidative phosphorylation, a process whose efficiency depends on the integrity of mitochondrial structure and function. However, with age, mitochondrial dysfunction inevitably occurs. From the perspective of free radical damage, the production of free radicals such as superoxide anions and hydrogen peroxide increases significantly during aging, while the level of endogenous antioxidants gradually declines. This makes mitochondrial DNA (mtDNA) highly susceptible to free radical attacks, leading to cumulative damage. Because mtDNA lacks the sophisticated error-checking mechanism of nuclear DNA, it is prone to mutations once damaged. These mutations interfere with the normal function of enzymes or carriers essential for mitochondrial oxidative metabolism, resulting in energy metabolism disorders. These disorders manifest as reduced activity of mitochondrial respiratory enzymes, decreased mitochondrial membrane potential, a significant reduction in ATP synthesis, and severe obstruction of oxidative phosphorylation. Simultaneously, changes in mitochondrial membrane potential disrupt the normal balance of calcium ion uptake and excretion, leading to intracellular calcium ion homeostasis imbalance and severely affecting normal cellular physiological functions. This mitochondrial dysfunction has a profound impact on the aging process. On the one hand, it is closely related to cellular senescence. Senescent cells are often accompanied by mitochondrial dysfunction. Excessive reactive oxygen species (ROS) produced by mitochondrial dysfunction can lead to DNA damage, activate related pathways, cause cell cycle arrest, and trigger cellular senescence. On the other hand, mitochondrial dysfunction also plays a key role in many age-related diseases, such as cancer, neurodegenerative diseases, metabolic diseases, and kidney diseases. Taking neurodegenerative diseases as an example, mitochondrial dysfunction leads to insufficient energy supply, which cannot meet the high energy demands of nerve cells. At the same time, the produced ROS can damage nerve cells, disrupt neurotransmitter metabolism, and thus lead to neuronal death and neurological dysfunction.

[0003] Although research on the role of mitochondrial dysfunction in aging has made some progress, current technologies still have many limitations. For example, the molecular mechanisms of mitochondrial dysfunction are not fully understood, and finding highly specific interventions to improve mitochondrial function is extremely difficult due to the highly complex and diverse phenotypes of mitochondrial dysfunction. Furthermore, in treating age-related diseases associated with mitochondrial dysfunction, existing treatments often fail to precisely target mitochondria, resulting in unsatisfactory therapeutic effects and potentially numerous side effects. Therefore, developing new technologies, methods, and substances that can effectively improve mitochondrial function and precisely intervene in the aging process related to mitochondrial dysfunction has become a critical issue that urgently needs to be addressed in this field.

[0004] Autophagy is a precisely regulated active process. When cells are stimulated by external factors, they maintain cellular homeostasis through a series of intracellular signal transduction processes. Autophagy is a continuous and dynamic process, mainly including autophagy induction, substrate recognition and selection, autophagosome formation and fusion with lysosomes, substrate degradation, and the release of degradation products into the cytoplasm. Many autophagy-related genes (ATGs) and their encoded proteins are involved in these processes. To date, more than 30 autophagy-related genes have been identified in eukaryotes, among which Atg1-10, Atg12-14, Atg16-18, Atg29, Atg31, and Atg101 are the main genes involved in autophagy. In the autophagy signaling pathway, silencing information reulator sirtuin 1 (SIRT1) is a deacetylase that catalyzes the removal of acetyl groups from many protein substrates, regulating autophagy through acetylation / deacetylation of forkhead box transcription factor O3 (FOXO3). Autophagy plays a crucial role in maintaining cellular homeostasis, responding to external stresses, and preventing disease. While autophagy helps slow age-related functional decline, cellular senescence is often accompanied by a decrease in autophagy levels, reduced ability to degrade damaged organelles and proteins, and accelerated aging. Furthermore, decreased autophagy levels can shorten lifespan due to gene dysfunction. Moderate activation of autophagy has anti-aging effects, and increasing autophagy levels can extend lifespan in many model organisms. In recent years, increasing research has shown that regulating autophagy can improve protein and mitochondrial homeostasis, thereby delaying organ function decline and extending lifespan.

[0005] Currently, although some drugs have been shown to regulate autophagy, existing drugs often suffer from poor specificity. This means that while regulating autophagy, they may have non-specific effects on other intracellular biological processes, leading to a range of side effects such as liver and kidney damage and immunosuppression. Furthermore, some drugs are unstable and prone to degradation during storage and transportation, resulting in drug inactivation. High research and development costs and complex manufacturing processes also limit the clinical application of these drugs. Even more challenging is the possibility that long-term or inappropriate use of these drugs may lead to drug resistance in cells, gradually reducing their efficacy.

[0006] Given the problems existing in the current technology, there is an urgent need to develop a safe, effective, and low-cost product for regulating autophagy and mitochondrial function, so as to provide new solutions for disease treatment, anti-aging and other fields. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction, along with its preparation method and applications. This invention first screens natural plant extracts with autophagy-regulating activity and those that improve mitochondrial dysfunction. Then, based on these natural plant extracts, other ingredients are scientifically and rationally combined to prepare a product that regulates eukaryotic autophagy and improves mitochondrial dysfunction, thereby alleviating aging safely and without side effects.

[0008] The technical solution of this invention is as follows:

[0009] A composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 10-30 parts broccoli pollen, 10-30 parts wheat germ microcapsule powder, 20-40 parts raspberry extract, 20-40 parts banana powder, 2-6 parts leucine, and 0.1-1 parts disodium pyrroloquinoline quinone.

[0010] According to a preferred embodiment of the present invention, the composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 15-25 parts broccoli pollen, 15-25 parts wheat germ microcapsule powder, 25-35 parts raspberry extract, 25-35 parts banana powder, 3-5 parts leucine, and 0.3-0.8 parts disodium pyrroloquinoline quinone.

[0011] According to a preferred embodiment of the present invention, the composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 20 parts broccoli pollen, 20 parts wheat germ microcapsule powder, 30 parts raspberry extract, 30 parts banana powder, 4 parts leucine, and 0.5 parts disodium pyrroloquinoline quinone.

[0012] According to a preferred embodiment of the present invention, the broccoli pollen is prepared according to the following method:

[0013] Cut fresh broccoli into 1cm pieces 3 After drying the broccoli into small pieces until the moisture content is less than or equal to 8 wt%, the dried broccoli is then pulverized and passed through an 80-mesh sieve to obtain broccoli pollen.

[0014] More preferably, the drying is carried out in an ultrasonic combined hot air drying oven, with the broccoli positioned directly below the ultrasonic probe, 5 mm away from it, the air velocity set at 2 m / s, the drying temperature at 70°C, and the ultrasonic intensity at 125.2 W / dm. 2 and 180.1W / dm 2 The ultrasonic mode is 5 seconds on and 5 seconds off.

[0015] According to a preferred embodiment of the present invention, the wheat germ microcapsule powder is prepared according to the following method:

[0016] Wheat is processed through a series of steps including cleaning, moistening, crushing, separating, drying, and enzyme inactivation to obtain wheat germ. The dried wheat germ is then pulverized and passed through an 80-mesh sieve to obtain wheat germ powder. The wheat germ powder is added to deionized water to obtain a mixture with a moisture content of 5-10%. The mixture is then puffed, dried to a moisture content of 2-6%, and after secondary pulverization and microwave sterilization, puffed wheat germ powder is obtained. Finally, wheat germ microcapsule powder is prepared by coating the puffed wheat germ powder with ethyl cellulose as the wall material.

[0017] A further preferred method involves using microwave drying to dry and inactivate the enzymes in the separated wheat germ. The processing capacity is 20 kg / h, the power is 4 kW, the time is 8 min, and the ventilation rate is 60 Nm. 3 / h, which deactivates the lipase and lipoxygenase contained in wheat germ, and the moisture content is ≤14.0%.

[0018] More preferably, the puffing is performed using a twin-screw extruder, which includes three puffing zones: zone one, zone two, and zone three. During puffing, zone one is closed, the temperature of zone two is 110–120°C, and the temperature of zone three is 120–140°C. The screw speed of the twin-screw extruder is 50 rpm, the feeding speed is 8–13 rpm, and the rotary cutting speed is 16–20 rpm.

[0019] More preferably, the particle size of the secondary pulverization is 80-100 mesh; the microwave sterilization conditions are: temperature 60-70℃, time 2-9 min.

[0020] More preferably, the coating method is bottom spraying, with the following specific parameters: air source pressure is 0.45 MPa, airtight pressure is 0.3 MPa, wall material flow rate is 2 mL / min, inlet air temperature is 50℃, outlet air temperature is 30℃, bed temperature is 40℃, and coating time is 120 min.

[0021] According to a preferred embodiment of the present invention, the raspberry extract is prepared according to the following method:

[0022] Fresh raspberries were dried to a moisture content of less than 10%, pulverized, and passed through a 40-mesh sieve to obtain raspberry powder. The raspberry powder was then immersed in 50% alcohol at a material-to-liquid ratio of 1:50 at 80°C for three extractions, each lasting 2.5 hours. The extracts were then combined. The extracts were concentrated at 65°C under a vacuum of 0.01 MPa for 50 minutes to obtain raspberry pulp. Finally, the raspberry pulp was spray-dried until the moisture content was less than or equal to 8 wt%, and passed through an 80-mesh sieve to obtain raspberry extract.

[0023] More preferably, the spray drying conditions are: inlet temperature 160-180℃, outlet temperature 65-75℃, peristaltic pump flow rate 45-55mL / min, and fan frequency 35-45Hz.

[0024] According to a preferred embodiment of the present invention, the banana powder is prepared according to the following method:

[0025] Peel and slice fresh bananas to obtain banana slices with a thickness of 0.3-0.6 cm; then soak the banana slices in a 0.5%-1% citric acid solution for 5 minutes, and then freeze-dry them under vacuum to obtain dried banana chips; finally, pulverize the dried banana chips and pass them through an 80-mesh sieve to obtain banana powder.

[0026] More preferably, the vacuum freeze-drying conditions are: vacuum degree of 1.0 Pa, freezing temperature of -58.7℃ to -59.3℃, and time of 26 hours.

[0027] The preparation method of the above-mentioned composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction includes the following steps:

[0028] (1) Broccoli pollen, wheat germ microcapsule powder, raspberry extract, banana powder, leucine, and disodium pyrroloquinoline quinone were passed through an 80-mesh sieve and then mixed evenly according to the ratio to obtain a mixture.

[0029] (2) Add water to the mixture to obtain a soft material; then granulate the soft material through a 20-mesh sieve, dry it at 37°C after granulation, and then granulate and sieve to obtain a composition that regulates eukaryotic cell autophagy and improves mitochondrial dysfunction.

[0030] The above-mentioned composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction is used in the preparation of anti-aging functional foods, pharmaceuticals or health products.

[0031] The active ingredients and their functions in this invention:

[0032] The broccoli pollen of this invention is rich in vitamins, minerals, and antioxidants, and is known as a "weight-loss meal" because it is relatively low in calories but provides abundant nutrition. Wheat germ is the most vigorous part of the wheat seed, rich in high-quality protein, fat, carbohydrates, various vitamins and minerals, as well as some physiologically active components needed by the human body.

[0033] Both broccoli pollen and wheat germ powder of this invention contain spermidine, a class of biogenic amines in the polyamine family. Polyamines are polycations that can interact with negatively charged molecules (including DNA, RNA, and lipids). Currently, induction of autophagy is considered the most important mechanism by which spermidine delays aging. Studies have found that spermidine can induce autophagy through the AKT / AMPK-FoxO3-Atg pathway, and can also promote the transport of histone deacetylase 4 (HDAC4) to the cell nucleus, reduce the content of cytoplasmic HDAC4, and thus enhance the acetylation and stability of microtubule-associated protein 1S (MAP1S) to activate autophagy. In addition, spermidine can act as an acetyltransferase inhibitor to regulate EP300 activity, thereby altering the acetylation status of autophagy-related proteins Atg5, Atg7, microtubule-associated protein 1 light 3 (LC3), and Atg12. Simultaneously, it can inhibit the phosphorylation of mammalian target of rapamycin (mTOR) and activate the phosphorylation of AMP-activated protein kinase (AMPK). AMPK inhibits mTOR at the functional level, which can further promote spermidine-induced autophagy. Therefore, broccoli pollen and wheat germ powder synergistically regulate cellular autophagy, thereby alleviating aging.

[0034] The raspberry extract of this invention contains abundant natural polyphenolic compound ellagitannin (ET), which is metabolized by intestinal flora to produce urolithin A (UA). UA activates mitophagy through both PINK1 / Parkin-dependent and PINK1 / Parkin-independent pathways. In the PINK1 / Parkin-dependent pathway, PINK1 protein accumulates on damaged mitochondria and recruits Parkin, which promotes ubiquitination of mitochondrial proteins. These ubiquitinated proteins are phosphorylated by PINK1 to form phosphorylated ubiquitin chains, attracting the autophagy adaptor protein LC3 and promoting the formation of autophagic vesicles and phagocytosis of mitochondria. In the PINK1 / Parkin-independent pathway, mitochondrial proteins such as BNIP3, NIX, and FUNDC1 directly interact with LC3, initiating the formation of autophagic vesicles and clearing damaged mitochondria. In summary, urolithin A regulates autophagy in eukaryotic cells through these two pathways, thereby playing a role in delaying aging and prolonging lifespan.

[0035] The banana powder of this invention is rich in various nutrients with potential health benefits: the pectin and phospholipids it contains may help maintain gastrointestinal health; the abundant glucose and fructose metabolism produce pyruvate, a key intermediate in the tricarboxylic acid cycle, which can provide energy for cellular mitochondria and exert an antioxidant effect by reducing the accumulation of reactive oxygen species (ROS). Furthermore, the polyphenols and tryptophan in the banana powder may also have a positive impact on mood regulation.

[0036] The leucine in this invention can regulate protein turnover in the body. This physiological process includes maintaining protein homeostasis, clearing aging and damaged proteins, synthesizing heat shock and immune-related proteins, participating in tissue repair and renewal, and responding to various nutritional and hormonal signals. When leucine levels are sufficient, it can promote protein synthesis in many organs such as muscles, liver, and small intestine. It can also activate the mammalian rapamycin protein (mTOR) pathway by binding to leucyl-tRNA. Upon activation, mTOR phosphorylates its downstream molecules S6K and 4EBP, a rate-limiting step in protein synthesis. Simultaneously, leucine can inhibit protein degradation in muscle cells and hepatocytes. Leucine supplementation can increase the expression of the pyruvate carrier MPC.

[0037] The pyrroloquinoline quinone disodium salt (PQQ) of this invention is an enzyme cofactor with redox activity, primarily involved in electron transport processes in the respiratory chain of microbial and mammalian cells. Studies have shown that PQQ exerts multiple biological effects by regulating mitochondrial homeostasis: it activates the PGC-1α signaling pathway, promoting mitochondrial DNA replication and new mitochondrial generation; it enhances ATP synthesis efficiency by stabilizing mitochondrial membrane potential; and it simultaneously activates mitophagy, selectively clearing dysfunctional mitochondria to achieve dynamic homeostasis. This dual regulatory mechanism gives PQQ significant advantages in improving cellular energy metabolism efficiency and delaying mitochondrial aging.

[0038] The beneficial effects of this invention are:

[0039] 1. The composition provided in this invention for regulating eukaryotic autophagy and improving mitochondrial dysfunction uses natural plant extracts: broccoli pollen, wheat germ microcapsule powder, and raspberry extract, as well as disodium pyrroloquinoline quinone (PQQ). Spermine in broccoli pollen and wheat germ powder induces autophagy by: 1) enhancing MAP1S; 2) altering the acetylation state of autophagy-related proteins Atg5, Atg7, microtubule-associated proteins 1LC3, and Atg12; and 3) inhibiting mTOR phosphorylation and activating AMPK. Urea in raspberry extract activates mitophagy through both PINK1 / Parkin-dependent and PINK1 / Parkin-independent pathways. PQQ regulation can restore mitochondrial health to alleviate damage to mitophagy. Simultaneously, PQQ activates the PGC-1α signaling pathway, promoting mitochondrial DNA replication and new mitochondrial generation; and by stabilizing mitochondrial membrane potential, it enhances ATP synthesis efficiency. These three substances synergistically provide energy to mitochondria and improve mitochondrial dysfunction. The above four components activate mitochondria through regulating proteins, PINK1 / Parkin-dependent pathways, and PINK1 / Parkin-independent pathways, thereby synergistically inducing autophagy.

[0040] 2. The composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction provided by this invention also contains banana powder and leucine. The banana powder produces pyruvate through sugar degradation. Pyruvate enters the mitochondria and generates ATP. During pyruvate transport, MPC allows pyruvate to enter the MPC's internal "channel" from the cytoplasm, where it is then released into the mitochondria. Leucine increases the expression of the pyruvate carrier MPC, thereby bringing more pyruvate into the mitochondria to generate energy. These two components enhance ATP production and improve mitochondrial dysfunction through the pyruvate-ATP pathway.

[0041] 3. In this invention, broccoli pollen is dried using a combination of ultrasonic technology and hot air drying to more effectively preserve its antioxidant components. Wheat germ powder is processed using microencapsulation technology to maximize the retention of its active ingredients. Raspberry extract is prepared using a spray-drying process, which helps retain more urolithin A. Banana powder undergoes citric acid color protection and freeze-drying processes to help retain more nutrients. Through these methods, this invention successfully maximizes the retention of the active ingredients of each raw material under the same addition conditions, thereby more effectively synergistically promoting autophagy, providing energy to mitochondria, improving mitochondrial dysfunction, and ultimately achieving an anti-aging effect.

[0042] 4. The composition provided by this invention for regulating eukaryotic autophagy and improving mitochondrial dysfunction combines the effects of the above six components. It is rationally formulated, safe and effective. Each component activates mitochondria by regulating proteins, the PINK1 / Parkin-dependent pathway, and the PINK1 / Parkin-independent pathway. At the same time, it promotes mitochondrial DNA replication and new mitochondrial generation by activating the PGC-1α signaling pathway, stabilizes mitochondrial membrane potential, and improves ATP synthesis efficiency. It synergistically activates mitophagy, improves mitochondrial dysfunction, and thus alleviates aging. Attached Figure Description

[0043] Figure 1 The results of laser confocal detection analysis in Experiment Example 1 are shown.

[0044] Figure 2 The results show the relative expression levels of SIRT1 mRNA in Experiment Example 1.

[0045] Figure 3 The results of laser confocal detection analysis in Experiment Example 2 are shown.

[0046] Figure 4 The results show the relative expression levels of SIRT1 mRNA in Experiment Example 2.

[0047] Figure 5 The results of ATP detection in Experiment Example 3.

[0048] Figure 6 The results of mitochondrial membrane potential detection in Experiment Example 3.

[0049] Figure 7 The results of ATP detection are shown in Experiment Example 4.

[0050] Figure 8 The results of mitochondrial membrane potential detection in Experiment Example 4. Detailed Implementation

[0051] The present invention will be described in detail below with reference to specific embodiments. These embodiments are merely illustrative and intended to facilitate understanding, implementation, or use of the technical solutions and methods of the present invention by those skilled in the art, and are not intended to limit the scope of protection of the present invention. Broccoli, wheat, raspberries, and bananas are available in major chain supermarkets in Jinan, Shandong. Disodium pyrroloquinoline quinone is available from Hubei Meiqi Health Technology Co., Ltd., and leucine is available from Shandong Pingju Biotechnology Co., Ltd.

[0052] In this example, broccoli pollen was prepared according to the following method:

[0053] 1) Select fresh, green broccoli and cut it into pieces about 1cm long. 3 Small pieces;

[0054] 2) Arrange the chopped broccoli florets evenly in the serving dish;

[0055] 3) Place the material tray in the ultrasonic combined hot air drying oven, with the broccoli pieces positioned directly below the ultrasonic probe, approximately 5mm away. Set the airflow speed to 2m / s, the drying temperature to 70℃, and the ultrasonic intensity to 125.2W / dm. 2 and 180.1W / dm 2 The ultrasonic mode is 5 seconds on and 5 seconds off, drying until the moisture content is less than or equal to 8 wt% (dry basis).

[0056] 4) Place the dried broccoli in a grinder and grind it through an 80-mesh sieve to obtain broccoli powder.

[0057] Wheat germ microcapsule powder is prepared according to the following method:

[0058] 1) Wheat cleaning: Wheat is cleaned by sequentially using a vibrating screen, destoner, gravity separator, magnetic separator, and fine separator to remove impurities, controlling impurities to ≤0.2% and magnetic metal content to ≤0.003g / kg;

[0059] 2) Wheat rinsing: The wheat is rinsed in sequence using a rinsing bin and a high-power watering machine. The rinsing time is controlled between 16 and 30 hours, and the net moisture content of the wheat is 14.0 to 16.55%.

[0060] 3) Crushing: The wheat is initially crushed by using a milling machine and a bran brushing machine in sequence to separate the wheat germ from the grain.

[0061] 4) Separation: The wheat germ and grains are separated in sequence using a purifier, a grinder, and a vibrating screen to obtain the wheat germ;

[0062] 5) Drying and enzyme inactivation: Microwave drying was used to dry and inactivate the enzymes in the isolated wheat germ. The processing capacity was 20 kg / h, the power was 4 kW, the time was 8 min, and the ventilation rate was 60 Nm. 3 / h, which deactivates the lipase and lipoxygenase contained in wheat germ, and the moisture content is ≤14.0%;

[0063] 6) Crushing: Crush the dried wheat germ and pass it through an 80-mesh sieve to obtain wheat germ powder.

[0064] 7) Extrusion: Wheat germ powder is added to deionized water to obtain a mixture with a moisture content of 5-10%. The mixture is then extruded using a twin-screw extruder, which consists of three extrusion zones: zone one, zone two, and zone three. During extrusion, zone one is closed, zone two is at a temperature of 110-120℃, and zone three is at a temperature of 120-140℃. The screw speed of the twin-screw extruder is 50 rpm, the feeding speed is 10 rpm, and the rotary cutting speed is 18 rpm. The extruded product is dried in a 65℃ oven for 40 minutes to reduce its moisture content to 5%. After secondary pulverization, the powder is passed through an 80-mesh sieve and then microwaved at 65℃ for 5 minutes to obtain wheat germ extruded powder.

[0065] 8) Using wheat germ puffed powder as the core material and 4.25% ethyl cellulose (solvent: anhydrous ethanol) as the wall material, the coating was carried out by bottom spraying. The specific parameters were: air source pressure of 0.45 MPa, airtight pressure of 0.3 MPa, wall material flow rate of 2 mL / min, inlet air temperature of 50℃, outlet air temperature of 30℃, bed temperature of 40℃, and coating time of 120 min, to obtain wheat germ microcapsule powder.

[0066] Raspberry extract was prepared according to the following method:

[0067] 1) Select fresh, disease-free raspberries that are conical or flattened conical in shape;

[0068] 2) Dry at low temperature to control its moisture content below 10%;

[0069] 3) Crush the dried raspberries and pass them through a 40-mesh sieve;

[0070] 4) Add the crushed raspberries to 50% alcohol at a material-to-liquid ratio of 1:50, at a temperature of 80℃, and extract three times, each time for 2.5 hours. Stir continuously during the extraction process to ensure that the raw materials are fully dissolved in the alcohol. After the extraction is completed, filter out the solid residue and combine the three extracts.

[0071] 5) Concentrate the extract at 65℃ and 0.01MPa vacuum for 50 min;

[0072] 6) The concentrated raspberry pulp was spray-dried at a homogenization pressure of 25 MPa, an inlet temperature of 160–180 °C, an outlet temperature of 70 °C, a peristaltic pump flow rate of 50 mL / min, and a fan frequency of 40 Hz. The pulp was dried until the moisture content was less than or equal to 8 wt% (dry basis), and then passed through an 80-mesh sieve to obtain the raspberry extract.

[0073] Banana powder is prepared according to the following method:

[0074] 1) Raw material selection: Select bananas that are 15cm in length, have no mechanical damage, and are 80% ripe as raw materials;

[0075] 2) Peel and slice: Peel and slice the selected bananas, keeping the slices about 0.5cm thick.

[0076] 3) Color preservation: Soak banana slices in a 0.5%-1% citric acid solution for 5 minutes;

[0077] 4) Drying: Place the processed banana slices in a prepared vacuum freeze dryer. The vacuum freeze drying conditions are: vacuum degree of 1.0 Pa, freezing temperature of -58.7℃ to -59.3℃, and time of 26 hours to obtain dehydrated banana slices.

[0078] 5) Grinding and sieving: Put the dehydrated banana slices into a high-speed grinder, turn on the high-speed grinder switch, and run the grinder at high speed until the banana slices are completely turned into fine powder particles. Then pour them into an 80-mesh stainless steel sieve. After sieving, you will get banana powder.

[0079] Example 1

[0080] A composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 20 parts broccoli pollen, 20 parts wheat germ microcapsule powder, 30 parts raspberry extract, 30 parts banana powder, 4 parts leucine, and 0.5 parts disodium pyrroloquinoline quinone.

[0081] The preparation method of the above-mentioned composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction includes the following steps:

[0082] (1) Broccoli pollen, wheat germ microcapsule powder, raspberry extract, banana powder, leucine, and disodium pyrroloquinoline quinone were passed through an 80-mesh sieve and then mixed evenly according to the ratio to obtain a mixture.

[0083] (2) Add water to the mixture to obtain a soft material; then granulate the soft material through a 20-mesh sieve, dry it at 37°C after granulation, and then granulate and sieve to obtain a composition that regulates eukaryotic cell autophagy and improves mitochondrial dysfunction.

[0084] Example 2

[0085] A composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 30 parts broccoli pollen, 30 parts wheat germ microcapsule powder, 20 parts raspberry extract, 20 parts banana powder, 6 parts leucine, and 0.1 parts disodium pyrroloquinoline quinone.

[0086] The specific preparation method is the same as in Example 1.

[0087] Example 3

[0088] A composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 10 parts broccoli pollen, 10 parts wheat germ microcapsule powder, 40 parts raspberry extract, 40 parts banana powder, 2 parts leucine, and 1 part disodium pyrroloquinoline quinone.

[0089] The specific preparation method is the same as in Example 1.

[0090] Comparative Example 1

[0091] A composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 35 parts broccoli pollen, 35 parts wheat germ microcapsule powder, 45 parts raspberry extract, 45 parts banana powder, 8 parts leucine, and 1.2 parts disodium pyrroloquinoline quinone.

[0092] The specific preparation method is the same as in Example 1.

[0093] Comparative Example 2

[0094] A composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction comprises the following raw materials in parts by weight: 5 parts broccoli pollen, 5 parts wheat germ microcapsule powder, 15 parts raspberry extract, 15 parts banana powder, 1 part leucine, and 0.05 parts disodium pyrroloquinoline quinone.

[0095] The specific preparation method is the same as in Example 1.

[0096] Comparative Example 3

[0097] A composition, free of raspberry extract, comprises the following ingredients in parts by weight: 20 parts broccoli pollen, 20 parts wheat germ microcapsule powder, 30 parts banana powder, 4 parts leucine, and 0.5 parts disodium pyrroloquinoline quinone.

[0098] The specific preparation method is the same as in Example 1.

[0099] Comparative Example 4

[0100] A composition that does not contain pyrroloquinoline quinone disodium salt, comprising the following parts by weight of raw materials: 20 parts broccoli pollen, 20 parts wheat germ microcapsule powder, 30 parts raspberry extract, 30 parts banana powder, and 4 parts leucine.

[0101] Comparative Example 5

[0102] A composition that does not contain broccoli pollen and wheat germ microcapsule powder, comprising the following parts by weight of raw materials: 30 parts raspberry extract, 30 parts banana powder, 4 parts leucine, and 0.5 parts disodium pyrroloquinoline quinone.

[0103] Comparative Example 6

[0104] A composition, free of banana powder, comprising the following parts by weight of raw materials: 20 parts broccoli pollen, 20 parts wheat germ microcapsule powder, 30 parts raspberry extract, 4 parts leucine, and 0.5 parts disodium pyrroloquinoline quinone.

[0105] Comparative Example 7

[0106] A composition, which does not contain leucine, comprises the following raw materials in parts by weight: 20 parts broccoli pollen, 20 parts wheat germ microcapsule powder, 30 parts raspberry extract, 30 parts banana powder, and 0.5 parts disodium pyrroloquinoline quinone.

[0107] Experimental Example 1

[0108] The compositions prepared in Examples 1-3 and Comparative Examples 1-2 were added to 100 mL of DMEM medium, with an amount of 4 g.

[0109] Rat cardiomyocytes H9c2 were cultured in DMEM medium containing 10% fetal bovine serum until the logarithmic growth phase, and then randomly divided into a blank control group and experimental groups 1–5. The blank control group was given an equal volume of DMEM medium, experimental groups 1–3 were given DMEM medium containing the compositions of Examples 1–3, and experimental groups 4–5 were given DMEM medium containing the compositions of Comparative Examples 1–2. Rat cardiomyocytes H9c2 were cultured in each medium for 72 hours.

[0110] 1. Immunofluorescence detection of intracellular LC3B protein expression levels

[0111] H9c2 rat cardiomyocytes from the blank control group and experimental groups 1-5 were placed in 24-well plates containing round glass slides. After fixation, washing, and blocking, the cells were incubated with primary antibody LC3 (1:1000) for 1.5 h, followed by incubation with fluorescently labeled secondary antibody (1:2000) at room temperature for 2 h. After washing, Hoechst 33342 dilution was added, and the cells were incubated at room temperature for 10 min. The cells were then observed and photographed using a laser confocal microscope. The results of the laser confocal microscopy analysis are as follows: Figure 1 As shown.

[0112] 2. RT-qPCR detection of relative SIRT1 mRNA expression levels

[0113] (1) Sample preparation

[0114] Cells from the blank control group and experimental groups 1-5 were collected, lysed, and RNA and miRNA were extracted from the cells. After concentration determination, cDNA was obtained by reverse transcription.

[0115] The SIRT1 primer sequence was designed using Oligo 7 software as follows:

[0116] Forward: SIRT1-1623F17:5'-TTGCCACCAACACCTCT-3';

[0117] Reverse: SIRT1-1898R19:5'-AGGCCAGCATTTTCTCACT-3'.

[0118] (2) Samples were loaded onto a real-time fluorescence kit (q-PCR amplification using SYBR Green I dye) to determine the relative expression level of SIRT1 mRNA. The results are as follows: Figure 2 As shown.

[0119] Reaction system: 10 μL 2×Master Mix, 0.4 μL forward primer (10 μM), 0.4 μL reverse primer (10 μM), 2 μL template cDNA, and nucleic acid-free water to a final volume of 20 μL.

[0120] Amplification program: Pre-denaturation, 95℃ for 3 min; Denaturation, 95℃ for 15 sec; Annealing, 60℃ for 30 sec; Extension, 72℃ for 45 sec, 40 cycles; Termination extension, 72℃ for 5 min; Final hold at 4℃.

[0121] Depend on Figure 1 It can be seen that, compared with the blank control group, the fluorescence intensity of LC3B in cells of experimental groups 1-3 was significantly enhanced, and the fluorescence enhancement intensity of experimental groups 1-3 decreased sequentially; the fluorescence intensity of LC3B in cells of experimental groups 4-5 was enhanced, but the enhancement effect was not as good as that of experimental groups 1-3, and the fluorescence enhancement intensity of LC3B in cells of experimental groups 4-5 decreased sequentially.

[0122] Depend on Figure 2 It was found that, compared with the blank control group, SIRT1 mRNA expression was significantly increased in cells 1-3 of the experimental group, and the expression showed a decreasing trend in that order, with significant differences (P<0.01). Although SIRT1 mRNA expression was also increased in cells 4-5 of the experimental group, there was no significant difference (P>0.05).

[0123] The above experimental results show that, compared with the compositions of Comparative Examples 1-2, the compositions of Examples 1-3 of the present invention have a more significant effect on regulating autophagy in cells.

[0124] Experimental Example 2

[0125] Based on the experimental results of Example 1, the composition prepared in Example 1 was added to 100 mL of DMEM culture medium at amounts of 2 g, 4 g, and 8 g, respectively, as low, medium, and high dosage groups. Then, the compositions prepared in Comparative Examples 3-5 were added to the DMEM culture medium at amounts of 4 g each.

[0126] Rat cardiomyocytes H9c2 were cultured in DMEM medium containing 10% fetal bovine serum until the logarithmic growth phase, and then divided into a blank control group and experimental groups 1-6. The blank control group was given an equal volume of DMEM medium, experimental groups 1-3 were given DMEM medium containing low, medium, and high doses of the composition of Example 1, respectively, and experimental groups 4-6 were given DMEM medium containing the compositions of Comparative Examples 3-5, respectively. Rat cardiomyocytes H9c2 were cultured in each medium for 72 hours.

[0127] 1. Following the method described in Experimental Example 1, the expression level of intracellular LC3B protein was detected by immunofluorescence. The results are as follows: Figure 3 As shown.

[0128] 2. Following the method described in Experiment Example 1, the relative expression level of SIRT1 mRNA was detected by RT-qPCR. The results are as follows: Figure 4 As shown.

[0129] Depend on Figure 3 It can be seen that, compared with the blank control group, the fluorescence intensity of LC3B in cells 1-3 of the experimental group was significantly enhanced, and the fluorescence intensity of cells 1-3 of the experimental group increased sequentially. Although the fluorescence intensity of LC3B in cells 4-6 of the experimental group was also enhanced, it was not significant.

[0130] Depend on Figure 4 It was found that, compared with the blank control group, SIRT1 mRNA expression was significantly increased in experimental groups 1-3, showing a sequential increasing trend, with significant differences (P<0.01). Although SIRT1 mRNA expression was also increased in cells of experimental groups 4-6, there was no significant difference (P>0.05).

[0131] The above data show that the absence of any one of the components such as broccoli pollen, wheat germ microcapsule powder, raspberry extract, and PQQ reduces the function of regulating autophagy, indicating that the broccoli pollen, wheat germ microcapsule powder, raspberry extract, and PQQ of the present invention synergistically regulate autophagy.

[0132] Experimental Example 3

[0133] Add 4g of the compositions prepared in Examples 1-3 and Comparative Examples 1-2 to 100ml of purified water.

[0134] Human neuroblastoma cells SH-SY5Y were passaged in DMEM / F12 medium + 15% fetal bovine serum + 1% PS at 37°C, 5% CO2, and saturated humidity. They were randomly divided into a blank control group, a high-glucose control group, and experimental groups 1–5. In the blank control group, SH-SY5Y cells were cultured normally for 24 h, while in the high-glucose control group, SH-SY5Y cells were co-cultured with 50 mM glucose for 24 h. Experimental groups 1–3 were treated with an aqueous solution containing the compositions of Examples 1–3 for 2 h, followed by co-culture with 50 mM glucose for 24 h. Experimental groups 4–5 were treated with an aqueous solution containing the compositions of Comparative Examples 1–2 for 2 h, followed by co-culture with 50 mM glucose for 24 h.

[0135] 1. ATP detection

[0136] After cell treatment, cells and supernatant from each group were collected. The procedure was followed according to the ATP kit instructions, and the results were analyzed using a microplate reader. The results are as follows: Figure 5 As shown.

[0137] 2. Mitochondrial membrane potential detection

[0138] After cell treatment, the cells were centrifuged (1000 rpm, 5 min), resuspended in 500 μL of 1×JC-1 staining solution, stained at 37℃ for 30 min, resuspended in 500 μL of PBS buffer, and passed through a 200-mesh sieve to prepare a single-cell suspension. Mitochondrial membrane potential was detected using flow cytometry. The results are as follows: Figure 6 As shown.

[0139] Depend on Figure 5 It can be seen that, compared with the blank control group, the ATP level in the high glucose control group was significantly decreased, while the ATP levels in experimental groups 1-3 were significantly increased compared with the high glucose control group (P < 0.01), and the degree of increase in ATP in experimental groups 1-3 decreased sequentially. The difference in ATP levels between experimental groups 4-5 and the high glucose control group was not significant (P > 0.05).

[0140] Depend on Figure 6 It was found that, compared with the blank control group, the membrane potential of the high-sugar control group was significantly increased, while the membrane potential of experimental groups 1-3 was significantly decreased compared with the high-sugar control group (P<0.01), and the degree of decrease in membrane potential of experimental groups 1-3 decreased sequentially. The difference in membrane potential between experimental groups 4-5 and the high-sugar control group was not significant (P>0.05).

[0141] The above experimental results show that, compared with the compositions of Comparative Examples 1-2, the compositions of Examples 1-3 of the present invention have a more significant effect in improving mitochondrial dysfunction.

[0142] Experiment Example 4

[0143] Based on the experimental results of Example 1, the composition prepared in Example 1 was added to purified water at amounts of 2g, 4g, and 8g, respectively, as low, medium, and high dosage groups. Then, the compositions prepared in Comparative Examples 4 and 6-7 were added to the purified water at an amount of 4g.

[0144] Human neuroblastoma cells SH-SY5Y were passaged in DMEM / F12 medium + 15% fetal bovine serum + 1% PS at 37°C, 5% CO2, and saturated humidity. They were randomly divided into a blank control group, a high-glucose control group, and experimental groups 1–6. In the blank control group, SH-SY5Y cells were cultured normally for 24 h; in the high-glucose control group, SH-SY5Y cells were co-cultured with 50 mM glucose for 24 h. Experimental groups 1–3 were treated with aqueous solutions containing low, medium, and high doses of the composition from Example 1 for 2 h, followed by co-culture with 50 mM glucose for 24 h. Experimental groups 4–6 were treated with aqueous solutions containing the compositions from Comparative Examples 4 and 6–7 for 2 h, followed by co-culture with 50 mM glucose for 24 h.

[0145] 1. Perform ATP detection according to the method described in Experiment Example 3. The results are as follows: Figure 7 As shown.

[0146] 2. Mitochondrial membrane potential was detected according to the method described in Experiment Example 1, and the results are as follows: Figure 8 As shown.

[0147] Depend on Figure 7 It can be seen that, compared with the blank control group, the ATP of the high glucose control group was significantly decreased, while the ATP of experimental groups 1-3 was significantly increased compared with the high glucose control group (P<0.01), and the degree of ATP increase in experimental groups 1-3 increased sequentially; there was no significant difference in ATP between experimental groups 4-6 and the high glucose control group (P>0.05).

[0148] Depend on Figure 8 It was found that, compared with the blank control group, the membrane potential of the high-glucose control group was significantly increased, while the membrane potential of experimental groups 1-3 was significantly decreased compared with the high-glucose control group (P<0.01), and the degree of decrease in membrane potential of experimental groups 1-3 increased sequentially. There was no significant difference in membrane potential between experimental groups 4-6 and the high-glucose control group (P>0.05).

[0149] The above data shows that the absence of any one of the three components—leucine, banana powder, and PQQ—can affect the improvement of mitochondrial function. The leucine, banana powder, and PQQ of this invention synergistically improve mitochondrial dysfunction.

[0150] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. A composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction, characterized in that, It is composed of the following raw materials in parts by weight: 10-30 parts broccoli pollen, 10-30 parts wheat germ microcapsule powder, 20-40 parts raspberry extract, 20-40 parts banana powder, 2-6 parts leucine, and 0.1-1 parts disodium pyrroloquinoline quinone. The raspberry extract was prepared according to the following method: Fresh raspberries were dried to a moisture content of less than 10%, pulverized, and passed through a 40-mesh sieve to obtain raspberry powder. The raspberry powder was then immersed in 50% alcohol at a material-to-liquid ratio of 1:50 at 80°C for three extractions, each lasting 2.5 hours. The extracts were then combined. The extracts were concentrated at 65°C under a vacuum of 0.01 MPa for 50 minutes to obtain raspberry pulp. Finally, the raspberry pulp was spray-dried until the moisture content was less than or equal to 8 wt%, and passed through an 80-mesh sieve to obtain raspberry extract. The spray drying conditions are as follows: inlet temperature 160~180℃, outlet temperature 70℃, peristaltic pump flow rate 50 mL / min, and fan frequency 40Hz.

2. The composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction as described in claim 1, characterized in that, It is composed of the following raw materials in parts by weight: 15-25 parts broccoli pollen, 15-25 parts wheat germ microcapsule powder, 25-35 parts raspberry extract, 25-35 parts banana powder, 3-5 parts leucine, and 0.3-0.8 parts disodium pyrroloquinoline quinone.

3. The composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction as described in claim 1, characterized in that, It is composed of the following ingredients in parts by weight: 20 parts broccoli pollen, 20 parts wheat germ microcapsule powder, 30 parts raspberry extract, 30 parts banana powder, 4 parts leucine, and 0.5 parts disodium pyrroloquinoline quinone.

4. The composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction as described in claim 1, characterized in that, The broccoli pollen was prepared according to the following method: Cut fresh broccoli into 1cm pieces 3 After drying the broccoli into small pieces until the moisture content is less than or equal to 8 wt%, the dried broccoli is then pulverized and passed through an 80-mesh sieve to obtain broccoli powder. The drying process was carried out in an ultrasonic combined hot air drying oven. The broccoli was positioned directly below the ultrasonic probe, 5 mm away from it. The airflow speed was set to 2 m / s, the drying temperature to 70°C, and the ultrasonic intensity to 125.2 W / dm. 2 and 180.1 W / dm 2 The ultrasonic mode is 5 seconds on and 5 seconds off.

5. The composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction as described in claim 1, characterized in that, The wheat germ microcapsule powder was prepared according to the following method: Wheat is processed through a series of steps including cleaning, moistening, crushing, separating, drying, and enzyme inactivation to obtain wheat germ. The dried wheat germ is then pulverized and passed through an 80-mesh sieve to obtain wheat germ powder. The wheat germ powder is added to deionized water to obtain a mixture with a moisture content of 5-10%. The mixture is then puffed, dried to a moisture content of 2-6%, and after secondary pulverization and microwave sterilization, puffed wheat germ powder is obtained. Finally, wheat germ microcapsule powder is prepared by coating the puffed wheat germ powder with ethyl cellulose as the wall material.

6. The composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction as described in claim 5, characterized in that, The enzyme inactivation process involves microwave drying of the isolated wheat germ, with a processing capacity of 20 kg / h, a power of 4 kW, a time of 8 min, and a ventilation rate of 60 Nm. 3 / h, which deactivates the lipases and lipoxygenases in wheat germ, and the moisture content is ≤14.0%; The puffing is performed using a twin-screw extruder, which includes three puffing zones: zone one, zone two, and zone three. During puffing, zone one is closed, the temperature in zone two is 110~120℃, and the temperature in zone three is 120~140℃. The screw speed of the twin-screw extruder is 50 rpm, the feeding speed is 8~13 rpm, and the rotary cutting speed is 16~20 rpm. The particle size of the secondary pulverization is 80-100 mesh; the microwave sterilization conditions are: temperature 60-70℃, time 2-9 min; The coating method is bottom spraying, with the following specific parameters: air source pressure is 0.45 MPa, airtight pressure is 0.3 MPa, wall material flow rate is 2 mL / min, inlet air temperature is 50℃, outlet air temperature is 30℃, bed temperature is 40℃, and coating time is 120 min.

7. The composition for regulating eukaryotic autophagy and improving mitochondrial dysfunction as described in claim 1, characterized in that, The banana powder is prepared according to the following method: Peel and slice fresh bananas to obtain banana slices with a thickness of 0.3-0.6 cm; then soak the banana slices in a 0.5%-1% citric acid solution for 5 minutes, and then freeze-dry them under vacuum to obtain dried banana chips; finally, pulverize the dried banana chips and pass them through an 80-mesh sieve to obtain banana powder. The vacuum freeze-drying conditions are as follows: vacuum degree is 1.0 Pa, freezing temperature is -58.7℃ to -59.3℃, and time is 26 h.

8. A method for preparing the composition according to any one of claims 1 to 7 for regulating eukaryotic autophagy and improving mitochondrial dysfunction, characterized in that, The steps include the following: (1) Broccoli pollen, wheat germ microcapsule powder, raspberry extract, banana powder, leucine, and disodium pyrroloquinoline quinone were passed through an 80-mesh sieve and then mixed evenly according to the ratio to obtain a mixture. (2) Add water to the mixture to obtain a soft material; then granulate the soft material through a 20-mesh sieve, dry it at 37°C after granulation, and then granulate and sieve to obtain a composition that regulates eukaryotic cell autophagy and improves mitochondrial dysfunction.

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